N-acetylhexanelactam

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-03-27 to 2017-04-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

other: (in chemico) reactivity against synthetic peptides with a thiol or amino group

Test material

Test material information

Constituent 1

Reference substance name:

N-acetylhexanelactam

EC Number:

217-565-6

EC Name:

N-acetylhexanelactam

Cas Number:

1888-91-1

Molecular formula:

C8H13NO2

IUPAC Name:

1-acetylazepan-2-one

Test material form:

liquid

Details on test material:

Batch: not yet known

Specific details on test material used for the study:

Name: N-acetylhexanelactam
Batch No.: ESD0024613
Chemical name: 1-acetylazepan-2-one
CAS No 1888-91-1
Purity 98.1% (GC)
Physical State: liquid
Molecular Weight: 155.2 g/mol
Colour: light yellow clear
Storage Conditions: room temperature
Stability: stable at storage conditions
Expiry Date: 06 May 2018
Safety Precautions: The routine hygienic procedures are sufficient to assure personnel health and safety.

In chemico test system

Details on the study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

Results and discussion

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean peptide depletion of the positive control for the cysteine peptide was between 60.8% and 100% (74.90 %). The mean peptide depletion of the positive control for the lysine peptide was between 40.2% and 69.0% (54.93%).

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%] cysteine run

Value:

3.82

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%] lysine run

Value:

19.69

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptance Criteria

The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the
positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control
replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine
percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine
percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

Any other information on results incl. tables

Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD7	0.0000	0.0000	0.0000	0.0000
STD6	153.8068	0.0167	139.6547	0.0167
STD5	321.6256	0.0334	281.4845	0.0334
STD4	642.2243	0.0667	555.3300	0.0667
STD3	1318.5444	0.1335	1100.1805	0.1335
STD2	2593.4377	0.2670	2176.1533	0.2670
STD1	5075.1016	0.5340	4280.0898	0.5340

 

Depletion of the Cysteine Peptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Concentration [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1193.1283	0.1239	74.99	74.90	0.25	0.34
	1187.7434	0.1233	75.10
	1210.7732	0.1258	74.62
Test Item	4595.2026	0.4810	3.67	3.82	0.21	5.37
	4592.3970	0.4807	3.73
	4577.0308	0.4791	4.05

Depletion of the Lysine Peptide

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Concentration [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1861.8433	0.2304	55.78	54.93	0.78	1.42
	1926.2322	0.2384	54.25
	1904.2300	0.2357	54.77
Test Item	3385.4136	0.4205	19.59	19.69	0.81	4.11
	3345.2500	0.4155	20.55
	3413.0500	0.4240	18.93

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model¹

Mean Cysteine andLysine PPD	ReactivityClass	DPRA Prediction²
0.00% ≤ PPD ≤ 6.38%	No or Minimal Reactivity	Negative
6.38% < PPD ≤ 22.62%	Low Reactivity	Positive
22.62% < PPD ≤ 42.47%	Moderate Reactivity
42.47% < PPD ≤ 100%	High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 prediction model¹

Cysteine PPD	ReactivityClass	DPRA Prediction²
0.00% ≤ PPD ≤ 13.89%	No or Minimal Reactivity	Negative
13.89% < PPD ≤ 23.09%	Low Reactivity	Positive
23.09% < PPD≤ 98.24%	Moderate Reactivity
98.24% < PPD ≤ 100%	High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

 

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In this study under the given conditions the test item showed low reactivity towards the peptides. The test item is considered to potentially have a sensitizing property but the data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

In the present study N-acetylhexanelactam was dissolved in acetonitrile. Based on a molecular weight of 155.2 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution.After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the test item samples. A slight precipitation was observed for standard 1 and the positive control samples. Since the acceptance criteria for the linearity of the standard curve as well as for the depletion range of the positive control were fulfilled, the observed precipitations were regarded as insignificant.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any sample.

No significant co-elution of test item with the peptide peaks was observed. Sensitizing potential of the test item was predicted from the mean peptide depletion of both peptides by comparing the peptide concentration of the test item samples to the corresponding reference control C (RC C).

In the lysine run a shift of the lysine peptide peak from 7.7 min (STD 1) to 7.0 (REF C2 ACN) up to 6.5 min (REF B6) was observed (see16.4.2). Since the reference control samples allow clear identification of the lysine peptide and since the test item did not elute between 6.5 and 7.7 min, that indeed the lysine peptide peak had shifted and that the peak area of that peak did only represent remaining lysine peptide. Furthermore, all validity criteria were fulfilled, and this shift was not considered having an influence on the quality or validity of the results.

The 100 mM stock solution of the test item showed low reactivity towards the synthetic peptides. The mean depletion of both peptides was >6.38% (11.75%). Based on the prediction model 1 the test item is considered to potentially have a sensitizing property.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.92%.